A green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, or a modified protein thereof, is capable of recombinant expression in heterologous cells, and in particular, in various types of mammalian cells. Moreover, the obtained recombinant protein exhibits fluorescent properties in host cells. With use of such features, the GFP from A. victoria and a homologue thereof have been used as in vivo fluorescent marker proteins capable of expressing in animal cells for various types of targets or intended uses in the fields of biochemistry, cell physiology, and medicine (See Reference 1: Lippincott-Schwartz, J., G. H. Patterson, Science, Vol. 300, 87-91 (2003); Reference 2: Tsien, R. Y., Annu. Rev. Biochem. Vol. 67, 509-544 (1998)).
With regard to the GFP from A. victoria, a mechanism required for exhibiting the fluorescent properties thereof has been studied. First, it has been revealed that in the process of the folding of the translated polypeptide of the GFP into its natural steric structure, it folds in the form of mature GFP having fluorescent properties through the steps of cyclization of its internal tripeptide site, from which the fluorescent moiety is constructed, and the subsequent oxidation thereof. Moreover, it has also been confirmed that SYG at the positions 65-67 in the deduced amino acid sequence of wild-type GFP from A. victoria is such an internal tripeptide site, from which the fluorescent moiety is formed. With regard to the wild-type GFP from A. victoria, the result of X-ray crystal structure analysis thereof has been published. In the three-dimensional structure thereof, 11 β strands constitute a barrel form, and one α helix is positioned in the center portion of the barrel form so as to pass through from the upper portion to the lower portion thereof. This form as a whole is referred to as can “β”. The SYG at the positions 65-67 exists in the α helix, and the fluorescent moiety formed therefrom is located almost in the center of the “β can”, so that it can be maintained in a hydrophobic environment isolated from the surrounding solvent molecules (water molecules) (See Reference 3: Ormo, M. et al., Science Vol. 273, 1392-1395 (1996); Reference 4: Yang, F. et al., Nature Biotech., Vol. 14, 1246-1251 (1996)).
It is assumed that a mechanism of conversion of the peptide into mature GFP, post to the translation, namely, such formation of a p-hydroxybenzolideneimidazolinone structure of the fluorescent moiety from the SYG existing in the α helix is carried out through the following process.                Folding post to translation (transition to distorted configuration)        
                Cyclization and dehydration steps        
                Oxidation step: completion of p-hydroxybenzylideneimidazolinone structure        

Finally, the fluorescent moiety, p-hydroxybenzylideneimidazolinone structure, is in a state of equilibrium between the following neutral form and ionized form.                Equilibrium between the ionized form and neutral form in p-hydroxybenzylideneimidazolinone structure        

Accordingly, with regard to the wild-type GFP from A. victoria, a peak at 395 to 397 nm of the maximum absorption wavelength corresponding to absorption of the neutral form and a peak at 470 to 475 nm of the maximum absorption wavelength corresponding to absorption of the ionized form are observed on an excitation spectrum. On the other hand, on a fluorescence spectrum, a peak at 504 nm of the maximum fluorescence wavelength corresponding to the fluorescence of the ionized form is observed. The 6-membered ring of “p-hydroxybenzyl” contained in the fluorescent moiety is coupled with the 5-membered ring of “imidazolinone” via an “idene” structure. If such a structure as a whole has a plane configuration, a π-electron conjugated system is expanded, and the energy difference between the lowest electronically excited state and the ground state is decreased. Specifically, since hyperconjugation of the aforementioned tautomeric form is present in the ionized form structure, the whole structure has a plane configuration. On the other hand, in the neutral form structure, if the 6-membered ring of “p-hydroxybenzyl” is slightly tilted from a plane on which the “idene” structure and the 5-membered ring of “imidazolinone” lie, the π-electron conjugated systems of both portions are divided, and light absorption is mainly observed from the ground state localized in the 6-membered ring of “p-hydroxybenzyl”.
Moreover, many attempts to modify the amino acid sequence of the wild-type GFP from A. victoria to change the fluorescence wavelength have been reported. Specifically, a way for introducing a mutation that influences on a π-electron conjugated system acting as the fluorescent moiety into the amino acid sequence, so that the resulted fluorescence is blue-shifted from the original green color to a blue or blue green color, or so that the resulted fluorescence is red-shifted to a yellow color, has been reported.
In the case of S65T-GFP formed by replacing Ser at position 65 with Thr in SYG at positions 65-67 that constitutes the fluorescent moiety, when TYG forms a fluorescent moiety in the same manner, the state of equilibrium between the neutral form and the ionized form is inclined to the ionized form. As a result, in S65T-GFP, a peak at 489 to 490 nm of the maximum absorption wavelength corresponding to absorption of the ionized form is mainly observed on an excitation spectrum. In addition, on a fluorescence spectrum, a peak at 510 to 511 nm of the maximum fluorescence wavelength corresponding to the fluorescence of an ionized form is measured, and thus it is somewhat red-shifted, when compared with the fluorescence of the wild-type GFP.
Even if a mutation is not introduced into SYG at positions 65-67 constituting a fluorescent moiety, when the folding into a natural steric structure is conducted as a means for promoting ionization of the phenolic hydroxyl group of Tyr, a method of introducing a mutation by replacing Glu at position 222 existing in two β strands (β10, β11) on the C-terminal side existing around the p-hydroxybenzylideneimidazolinone structure as a fluorescent moiety with Gly has been reported, for example. That is to say, if a carboxyl group on the side chain of Glu at position 222 that functions as a proton donator is eliminated, the phenolic hydroxyl group of Tyr makes up for such an eliminated carboxyl group. As a result, ionization of the phenolic hydroxyl group of Tyr is promoted.
Although the “idene” structure and the 5-membered ring portion of “imidazolinone” included in the p-hydroxybenzylideneimidazolinone structure that constitutes the aforementioned fluorescent moiety of GFP are basically employed in the same manner, an attempt to replace a p-hydroxyphanyl group (phenol ring) from the side chain of Tyr at position 66 with a imidazole ring derived from His or a indole ring derived from Trp, so as to change the fluorescence wavelength of the wild-type GFP, has also been made.
In the case of Y66H-GFP wherein Tyr at position 66 is replaced with His, for example, it has been reported that its fluorescence is blue-shifted from the green fluorescence of the wild-type GFP and that it exhibits a blue fluorescence at 447 nm of the maximum wavelength. This Y66H-GFP having a fluorescent moiety formed from SHG is also referred to as BFP (Blue Fluorescent Protein). The fluorescent moiety in this BFP has a 1H-imiazol-4-yl-methylideneimidazolinone structure, in which the p-hydroxyphanyl group (phenol ring) derived from Tyr is changed with an imidazole ring derived from His. It is likely that such a 1H-imiazol-4-yl-methylideneimidazolinone structure is in a state of equilibrium between the following ionized form and neutral form.                Equilibrium between the ionized form and neutral form in 1H-imidazol-4-yl-methylideneimidazolinone structure        

In either case, due to the difference in π-electron conjugated systems, a peak at 383 nm of the maximum absorption wavelength is measured on its excitation spectrum, and a peak at 447 nm of the maximum fluorescence wavelength is measured on its fluorescence spectrum. The two peaks exhibit a blue shift.
On the other hand, in the case of Y66W-GFP wherein Tyr at position 66 is replaced with Trp, it has been reported that its fluorescence is blue-shifted from the green fluorescence of the wild-type GFP, and that it exhibits a blue green fluorescence at 485 nm of the maximum wavelength. This Y66W-GFP having a fluorescent moiety formed from SWG is also referred to as CFP (Cyan Fluorescent Protein). In the fluorescent moiety in this CFP, p-hydroxyphanyl group (phenol ring) derived from Tyr is replaced with an indole ring derived from Trp, and it has an indole-3-yl-methylideneimidazolinone structure.                Indole-3-yl methylideneimidazolinone structure        

The fluorescence spectrum of the aforementioned CFP shows a form obtained by overlapping two fluorescence peaks having only a small energy difference. Even on the corresponding excitation spectrum, a form obtained by overlapping two peaks having a small energy difference is shown. As a factor for giving such two types of peaks, it has been suggested that two fluorescent states (photoexcited states) coexist and that a certain state of equilibrium exists between the two fluorescent states (photoexcited states).
With regard to such BFP and CFP, in addition to the way for changing an aromatic ring group constituting a π-electron conjugated system itself by replacing Tyr at position 66 that forms a fluorescence moiety with another aromatic amino acid residue, a technique for red-shifting a fluorescence wavelength thereof by interaction (π-π-stacking) that is due to the overlapping of the π-electrons of both substances caused by the overlapping of plurality of the aromatic ring groups has also been reported. Specifically, when the folding into a natural steric structure is conducted, if Thr at position 203 existing in a β strand (β10) on the C-terminal side existing around the p-hydroxybenzylideneimidazolinone structure as a fluorescent moiety is replaced with an aromatic amino acid residue, an aromatic ring group from said aromatic amino acid residue is overlapped with the π-electron conjugated system of the indole-3-yl-methylideneimidazolinone structure, and in particular, with a p-hydroxyphanyl group (phenol ring) portion derived from Tyr. In the case of a modified form obtained by replacing Thr at position 203 with Tyr, for example, a peak around 516 nm of the maximum absorption wavelength is measured on an excitation spectrum, whereas a peak around 529 nm of the maximum fluorescence wavelength is measured on a fluorescence spectrum. The two peaks exhibit a red shift. A modified protein exhibiting a yellow fluorescence as a result of this type of π-π-stacking is named as YFP (Yellow Fluorescent Protein) (See Reference 5: Cubitt, A. B. et al., Trends Biochem. Sci., Vol. 20, 448-455 (1995)).
Aside from the aforementioned GFP from A. victoria and modified proteins thereof, a fluorescent protein from Discosoma striata has been known. In the case of this fluorescent protein, a peak around 558 nm of the maximum absorption wavelength is measured on an excitation spectrum, whereas a peak around 583 nm of the maximum fluorescence wavelength is measured on a fluorescence spectrum. That is to say, this is a fluorescent protein exhibiting a red fluorescence, and it is referred to as DsRFP, which indicates a red fluorescent protein from Discosoma striata. The fluorescent moiety of this DsRFP is formed from QYG. In the DsRFP, in addition to the p-hydroxybenzylideneimidazolinone structure, a region including the amide bond on the N-terminal side of Glu at position 65 functions as a fluorescent moiety. As a result, the aforementioned great red shift has been achieved (See Reference 6: Matz, M. V. et al., Nature Biotech., Vol. 17, 969-973 (1999); Reference 7: Verkhusha, et al., Nature Biotech., Vol. 22, 289-296 (2004)).    Non-Patent Document 1: Lippincott-Schwartz, J. G. H. Patterson, Science Vol. 300, 87-91 (2003)    Non-Patent Document 2: Tsien, R. Y., Annu. Rev. Biochem. Vol. 67, 509-544 (1998)    Non-Patent Document 3: Ormo, M. et al., Science Vol. 273, 1392-1395 (1996)    Non-Patent Document 4: Yang, F. et al., Nature Biotech., Vol. 14, 1246-1251 (1996)    Non-Patent Document 5: Cubitt, A. B. et al., Trends Biochem. Sci., Vol. 20, 448-455 (1995)    Non-Patent Document 6: Matz, M. V. et al., Nature Biotech., Vol. 17, 969-973 (1999)    Non-Patent Document 7: Verkhusha, et al., Nature Biotech., Vol. 22, 289-296 (2004)